Hypovirulence of Colletotrichum gloesporioides Associated with dsRNA Mycovirus Isolated from a Mango Orchard in Thailand
Round 1
Reviewer 1 Report
Aditya R. Suharto et al reported a dsRNA mycovirus genome feature and their potential biological function in Colletotrichum gloeosporioides. Authors confirmed that CgRV2 is related to hypovirulence and have potential virocontrol. Authors got lots of results and supplied some new information on mycovirus, so I recommend publishing in Viruses. Here I have a comment, authors could consider them.
CgRV2 is very close to CgRV1 with more than 90% identity at RDRP, I think CgRV1 and CgRV2 should belong to the same species, and they are different strains. Therefore, the name of “CgRV2” should be re-considered.
Author Response
According to your suggestion, we agree that they belong to the same species with different strains. So, we re-considered changing the name of mycovirus from Colletotrichum gloeosporioides RNA virus 2 to Colletotrichum gloeosporioides RNA virus 1 strain Ssa-44.1 (CgRV1-Ssa-44.1).
Here, I attached the revised version of the MS.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Revised version attached. Red highlights deleted text. Yellow highlights inserted text.
Comments for author File: Comments.pdf
Author Response
We have made changes according to your comments.
Author Response File: Author Response.pdf
Reviewer 3 Report
The article of viruses-1847547 described the identification of a mycovirus from the phytopathogen Colletotrichum gloeosporioides through dsRNA extraction, genome cloning, sequence and phylogenetic analyses. In addition, the virus was confirmed to be a hypovirulence associated mycovirus according to the virus-curing and biological analysis. The content of this article could be of potential interest as it might expand our understanding of virus distribution and their effects on the important pathogenic fungus C. gloeosporioides. However, some concerns should be addressed.
1. English writing needs to be improved because there are many small mistakes in the whole text.
2.For the fungus identification，the ITS is not sufficient to identify the pathogen to the species, although mango anthracnose is mainly caused by C. gloeosporioides.
3.The phylogenetic tree presented in the text was generated based on the ORF2 encoded RdRp, so the phylogenetic tree in the Figure S3 should be based on ORF1 rather than ORF2.
4.In 2.6.2, the vertical transmission rate of the virus in C. gloeosporioides strain Ssa-44.1 was measured as three replicates, however, this was showed as five replications in figure 4C. Be careful to be consistent.
5.What concerned me most is the taxonomic status of the virus CgRV2 in this study, which is 88.22 % and 90.19 % similar to the ORF1 and ORF2 encoded amino acids of CgRV2, respectively. Considering that CgRV1 and CgRV2 shared the same host and more than 90% RdRp similarity, it may not be appropriate to classify them as distinct viral species.
6.For biological properties confirmation, the authors only compared the biological characteristics of the original strain Ssa-44.1 with those of the virus eliminated strain Ssa-44.1#18. Strictly speaking, the original strain, the eliminated strain, and the horizontally transferred virulent strain should be compared together.
7.Only 17.60 + 2.08 % of the spores contained the virus, which should be considered to have low transfer efficiency. Therefore, this should be modified in line 352.
8.Sequences of the Colletotrichum gloeosporioides RNA virus 2 should be provided as supplementary material if the information is unavailable in the database at present.
Author Response
The article of viruses-1847547 described the identification of a mycovirus from the phytopathogen Colletotrichum gloeosporioides through dsRNA extraction, genome cloning, sequence and phylogenetic analyses. In addition, the virus was confirmed to be a hypovirulence associated mycovirus according to the virus-curing and biological analysis. The content of this article could be of potential interest as it might expand our understanding of virus distribution and their effects on the important pathogenic fungus C. gloeosporioides. However, some concerns should be addressed.
- English writing needs to be improved because there are many small mistakes in the whole text.
We have already arranged to send the article for proofreading. https://proof-reading-services.filemail.com/t/uFFlrhKc
- For the fungus identification, the ITS is not sufficient to identify the pathogen to the species, although mango anthracnose is mainly caused by gloeosporioides.
We do agree with the comment. We used morphological analysis, colony and conidia, together with ITS identification. Results confirmed that strain Ssa-44.1 identify as C. gloeosporioides. The data of conidia were added at the line 235-237. Actually, we already sent the genome for the draft whole genome sequencing, the data confirm that Ssa-44.1 is C. gloeosporioides. However, we have plan to publish the draft WGS in other research article. So, we can’t include the data here. But I still believe that the data of morphology, conidia and ITS sequence are enough to confirm that it is C. gloeosporioides.
- The phylogenetic tree presented in the text was generated based on the ORF2 encoded RdRp, so the phylogenetic tree in the Figure S3 should be based on ORF1 rather than ORF2.
Edited to ORF 1
- In 2.6.2, the vertical transmission rate of the virus in gloeosporioides strain Ssa-44.1 was measured as three replicates, however, this was showed as five replications in figure 4C. Be careful to be consistent.
Edited to 5 replicates. (Line 660)
- What concerned me most is the taxonomic status of the virus CgRV2 in this study, which is 88.22 % and 90.19 % similar to the ORF1 and ORF2 encoded amino acids of CgRV2, respectively. Considering that CgRV1 and CgRV2 shared the same host and more than 90% RdRp similarity, it may not be appropriate toclassify them as distinct viral species.
According to your suggestion, we do agree with you that they are belong to the same species with different strain. So, we re-considered to change the name of mycovirus from Colletotrichum gloeosporioides RNA virus 2 to Colletotrichum gloeosporioides RNA virus 1 strain Ssa-44.1 (CgRV1-Ssa44.1).
- For biological properties confirmation, the authors only compared the biological characteristics of the original strain Ssa-44.1 with those of the virus eliminated strain Ssa-44.1#18. Strictly speaking, the original strain, the eliminated strain, and the horizontally transferred virulent strain should be compared together.
The result was added to the Fig. 4.
- Only 17.60 + 2.08 % of the spores contained the virus, which should be considered to have low transfer efficiency. Therefore, this should be modified in line 352.
Edited in abstract and line 2222.
- Sequences of the Colletotrichum gloeosporioides RNA virus 2 should be provided as supplementary material if the information is unavailable in the database at present.
Already added in Suplementary Figure S5.
Author Response File: Author Response.pdf
Reviewer 4 Report
Line 81： “an anthracnose-infected mango leaves”
Line 239：Can authors provide the result of DNase I and S1 nuclease digestion? I think this result is important for a dsRNA virus.
Line 328: For that part, authors just provided the effect of CgRV2 on host growth and morphology, other than on pathogenicity, So the “hypovirulence” is not appropriate.
Fig.5: (1) Can authors provide bars for Fig.5A, B and C? (2) In Fig.5C, can authors provide another evidence that the pathogenicity test of SSa-44.1 and SSa-44.1#18 on same sample? It will help us to determine the difference on pathogenicity between these two strains.
Author Response
Line 81： “an anthracnose-infected mango leaves”
Edited to leaf.
Line 239：Can authors provide the result of DNase I and S1 nuclease digestion? I think this result is important for a dsRNA virus.
Already added in Suplementary Figure S2.
Line 328: For that part, authors just provided the effect of CgRV2 on host growth and morphology, other than on pathogenicity, So the “hypovirulence” is not appropriate.
Edited
Fig.5: (1) Can authors provide bars for Fig.5A, B and C? (2) In Fig.5C, can authors provide another evidence that the pathogenicity test of SSa-44.1 and SSa-44.1#18 on same sample? It will help us to determine the difference on pathogenicity between these two strains.
We already added the scale bar in fig 5. We don’t have the pathogenicity test of virus infected and virus free on the same mango. However, the Fig S6., Apple assay, we did show the results from the same apple fruit.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Accept after proof reading service should be fine.
Author Response
This edited version was sent for proofreading at Proof-Reading-Service.com
Reviewer 4 Report
Figure 5. I think the pathogenicity test should be performed on the same sample, otherwise we cannot compare the difference on pathogenicity between these two strains.
Fugure S6A. It is hard to see the Apple assay was performed on the same apple fruit, and the bar should be added in S6A.
Author Response
1. Figure 5. I think the pathogenicity test should be performed on the same sample, otherwise we cannot compare the difference on pathogenicity between these two strains.
I disagree that just the same fruit should be used to evaluate the pathogenicity test. With my expertise and understanding, I believe we can test it on many fruits. This can be published in a variety of journals.
For example,
Konsue, W.; Dethoup, T.; Limtong, S. Biological Control of Fruit Rot and Anthracnose of Postharvest Mango by Antagonistic Yeasts from Economic Crops Leaves. Microorganisms 2020, 8, 317. https://doi.org/10.3390/microorganisms8030317
Rungjindamai, N. (2016). Isolation and evaluation of biocontrol agents in controlling anthracnose disease of mango in Thailand. Journal of Plant Protection Research, 56, 306-311.
Danh, L.T.; Giao, B.T.; Duong, C.T.; Nga, N.T.T.; Tien, D.T.K.; Tuan, N.T.; Huong, B.T.C.; Nhan, T.C.; Trang, D.T.X. Use of Essential Oils for the Control of Anthracnose Disease Caused by Colletotrichum acutatum on Post-Harvest Mangoes of Cat Hoa Loc Variety. Membranes 2021, 11, 719. https://doi.org/10.3390/membranes11090719
LiPing Wang, JingJing Jiang, YanFen Wang, Ni Hong, Fangpeng Zhang, WenXing Xu and GuoPing Wang Hypovirulence of the Phytopathogenic Fungus Botryosphaeria dothidea: Association with a Coinfecting Chrysovirus and a Partitivirus J. Virol. 2014, 88(13):7517 . DOI: 10.1128/JVI.00538-14. Published Ahead of Print 23 April 2014.
Niu Y, Yuan Y, Mao J, et al. Characterization of two novel mycoviruses from Penicillium digitatum and the related fungicide resistance analysis. Scientific Reports. 2018 Apr;8(1):5513. DOI: 10.1038/s41598-018-23807-3. PMID: 29615698; PMCID: PMC5882929.
Li H, Bian R, Liu Q, Yang L, Pang T, Salaipeth L, Andika IB, Kondo H and Sun L (2019) Identification of a Novel Hypovirulence-Inducing Hypovirus From Alternaria alternata. Front. Microbiol. 10:1076. doi: 10.3389/fmicb.2019.01076
Yang S, Dai R, Salaipeth L, Huang L, Liu J, Andika IB and Sun L (2021) Infection of Two Heterologous Mycoviruses Reduces the Virulence of Valsa mali, a Fungal Agent of Apple Valsa Canker Disease. Front. Microbiol. 12:659210. doi: 10.3389/fmicb.2021.659210
2. Fugure S6A. It is hard to see the Apple assay was performed on the same apple fruit, and the bar should be added in S6A.
There is now a scale bar in the figure. I'm not sure how to snap a picture of an apple that makes it look symmetrical.
